Extracellular potential measuring device and method for fabricating the same

ABSTRACT

A device for measuring an extracellular potential of a test cell includes a substrate having a well formed in a first surface thereof and a first trap hole formed therein. The well has a bottom. The first trap hole includes a first opening formed in the bottom of the well and extending toward a second face of the substrate, a first hollow section communicating with the first opening via a first connecting portion, and a second opening extending reaching the second surface and communicating with the first hollow section via a second connecting portion. The first connecting portion has a diameter smaller than a maximum diameter of the first hollow section, greater than a diameter of the second connecting portion, and smaller than a diameter of the test cell. The device can retain the test cell securely and accept chemicals and the test cell to be put into the device easily.

TECHNICAL FIELD

[0001] The present invention relates to a device for evaluating abiosample, such as a cell, easily and fast by measuring an extracellularpotential an electrochemical change generated by the biosample. Thepresent invention also relates to a method of manufacturing the device.

BACKGROUND ART

[0002] Drugs are generally screened according to electrical activitiesof the cell as an index by a patch clamp method or a method usingchemical, such as fluorochrome or luminescence indicator. In the patchclamp method, a micro-electrode probe electrically records an iontransportation through a single channel of protein molecule at amicro-section called “patch” of cell membrane attached to a tip of amicropipet. This method is one of the few methods that can evaluatefunctions of a protein molecule in real time. (Refer to “MolecularBiology of the Cell” third edition by Garland Publishing Inc. New York.1994, written by Bruce Alberts et al. Japanese Edition “MolecularBiology of the Cell” pages 181-182, published from Kyouikusha Inc. 1995)

[0003] Fluorochrome or luminescence indicator which emits light inresponse to a change of a density of specific ion monitors migration ofthe ion in a cell, thereby measuring the electrical activities of thecell.

[0004] The patch clamp method requires expertise for producing andoperating the micropipet, and a long time to measure one sample, thusnot being suitable for screening a large number of chemical-compoundcandidates. The method using the fluorochrome or the like can screen alarge number of chemical-compounds candidates fast, but requires dyeingcells. A background of the cells may be colored due to pigment inmeasuring, and is decolorized according to lapse of time, thus reducingan S/N ratio.

SUMMARY OF THE INVENTION

[0005] A device for measuring an extracellular potential of a test cellincludes a substrate having a well formed in a first surface thereof anda first trap hole formed therein. The well has a bottom. The first traphole includes a first opening formed in the bottom of the well andextending toward a second face of the substrate, a first hollow sectioncommunicating with the first opening via a first connecting portion, anda second opening extending reaching the second surface and communicatingwith the first hollow section via a second connecting portion. The firstconnecting portion has a diameter smaller than a maximum diameter of thefirst hollow section, greater than a diameter of the second connectingportion, and smaller than a diameter of the test cell.

[0006] The device can retain the test cell securely and accept chemicalsand the test cell to be put into the device easily.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007]FIG. 1 is a perspective view of a device for measuring anextracellular potential in accordance with Exemplary Embodiment 1 of thepresent invention.

[0008]FIG. 2 is a sectional view of the device in accordance withEmbodiment 1.

[0009]FIG. 3 is an enlarged sectional view of the device in accordancewith Embodiment 1.

[0010]FIG. 4 is a sectional view of the device in accordance withEmbodiment 1 for illustrating its usage.

[0011]FIG. 5 is an enlarged sectional view of the device in accordancewith Embodiment 1.

[0012]FIG. 6 is an enlarged sectional view of the device in accordancewith Embodiment 1.

[0013]FIG. 7 is an enlarged sectional view of the device in accordancewith Embodiment 1.

[0014]FIG. 8 is an enlarged sectional view of the device in accordancewith Embodiment 1.

[0015]FIG. 9 is an enlarged sectional view of the device in accordancewith Embodiment 1.

[0016]FIG. 10 is a sectional view of the device in accordance withEmbodiment 1 for illustrating a method of manufacturing the device.

[0017]FIG. 11 is a sectional view of the device in accordance withEmbodiment. 1 for illustrating the method.

[0018]FIG. 12 is a sectional view of the device in accordance withEmbodiment. 1 for illustrating the method.

[0019]FIG. 13 is an enlarged sectional of the device in accordance withEmbodiment 1 for. illustrating the method.

[0020]FIG. 14 is an enlarged sectional of the device in accordance withEmbodiment 1 for. illustrating the method.

[0021]FIG. 15 is an enlarged sectional of the device in accordance withEmbodiment 1 for. illustrating the method.

[0022]FIG. 16 is an enlarged sectional of the device in accordance withEmbodiment 1 for. illustrating the method.

[0023]FIG. 17 is an enlarged sectional of the device in accordance withEmbodiment 1 for. illustrating the method.

[0024]FIG. 18 is an enlarged sectional of the device in accordance withEmbodiment 1 for. illustrating the method.

[0025]FIG. 19 is an enlarged sectional of the device in accordance withEmbodiment 1 for. illustrating the method.

[0026]FIG. 20 is an enlarged sectional view of the device in accordancewith Embodiment. 1 for illustrating another method of manufacturing thedevice.

[0027]FIG. 21 is an enlarged sectional view of the device in accordancewith Embodiment. 1 for illustrating another method.

[0028]FIG. 22 is an enlarged sectional view of the device in accordancewith Embodiment. 1 for illustrating another method.

[0029]FIG. 23 is a perspective view of a device for measuring anextracellular potential in accordance with Exemplary Embodiment 2 of theinvention.

[0030]FIG. 24 is a sectional view of the device in accordance withEmbodiment 2.

[0031]FIG. 25 is an enlarged sectional view of the device in accordancewith Embodiment 2.

[0032]FIG. 26 is a sectional view of the device in accordance withEmbodiment. 2 for illustrating a method of manufacturing the device.

[0033]FIG. 27 is a sectional view of the device in accordance withEmbodiment. 2 for illustrating the method.

[0034]FIG. 28 is a sectional view of the device in accordance withEmbodiment. 2 for illustrating the method.

[0035]FIG. 29 is a sectional view of another device for measuring anextracellular potential in accordance with Embodiment 1.

[0036]FIG. 30 is a perspective view of the device for measuring anextracellular potential in accordance with Exemplary Embodiment 3 of theinvention.

[0037]FIG. 31A is a sectional view of the device in accordance withEmbodiment 3.

[0038]FIG. 31B is an enlarged sectional view of the device in accordancewith Embodiment 3.

[0039]FIG. 32 is a sectional view of the device in accordance withEmbodiment 3 for. illustrating an operation of the device.

[0040]FIG. 33 is an enlarged sectional view of the device in accordancewith mbodiment 3.

[0041]FIG. 34 is an enlarged sectional view of the device in accordancewith Embodiment 3.

[0042]FIG. 35 is a sectional view of the device in accordance withEmbodiment 3 for. illustrating a method of manufacturing the device.

[0043]FIG. 36 is an enlarged sectional view of the device in accordancewith Embodiment 3 for illustrating the method.

[0044]FIG. 37 is an enlarged sectional view of the device in accordancewith Embodiment 3 for illustrating the method.

[0045]FIG. 38 is a sectional view of the device in accordance Embodiment3 for illustrating the method.

[0046]FIG. 39 is a sectional view of the device in accordance Embodiment3 for illustrating the method.

[0047]FIG. 40 is a sectional view of the device in accordance Embodiment3 for illustrating the method.

[0048]FIG. 41 is a sectional view of the device in accordance Embodiment3 for illustrating the method.

[0049]FIG. 42 is a sectional view of the device in accordance Embodiment3 for illustrating the method.

[0050]FIG. 43 is a sectional view of a device for mesuring anextracellular potential in accordance with Exemplary Embodiment 4 of theinvention.

[0051]FIG. 44 is an enlarged sectional view the device in accordancewith Embodiment 4 for illustrating a method of manufacturing the device.

[0052]FIG. 45 is an enlarged sectional view of the device in accordancewith Embodiment 4 for illustrating the method.

[0053]FIG. 46 is an enlarged sectional view of a device for measuring anextracellular potential in accordance with Exemplary Embodiment 5 of theinvention for illustrating a method of manufacturing the device.

[0054]FIG. 47 is an enlarged sectional view of the device in accordancewith Embodiment 5 for illustrating the method.

[0055]FIG. 48 is an enlarged sectional view of the device in accordancewith Embodiment 5 for illustrating the method.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0056] Exemplary Embodiment 1

[0057]FIG. 1 is a perspective view of a device for measuring anextracellular potential in accordance with Exemplary Embodiment of thepresent invention. FIG. 2 is a sectional view of the device. FIG. 3 isan enlarged sectional view of the device.

[0058] The measuring device includes substrate 1 made of silicon havingwell 2 formed therein. Bottom 3 of well 2 has plural trap-holes 101formed therein for retaining cells. Each of trap-holes 101 includesfirst opening 4, hollow section 6, and second opening 5 aligned in thisorder on a straight line. A diameter of first opening 4 is smaller thana maximum diameter of hollow section 6, and greater than a diameter ofsecond opening 5.

[0059] Respective specific sizes of those portions are optimallydetermined according to a size of a test cell. For a test cell having adiameter of 25 μm, for instance, first opening 4 has a diameter of 20μm, which is smaller than 25 μm, and hollow section 6 has the maximumdiameter of 35 μm, which is greater than that of the cell. A diameter ofsecond opening 5 is determined to be about 10 μm, which is smaller thanthat of the test cell. A test cell generally has a diameter ranging fromseveral micrometers to several tens micrometers. Therefore, the diameterof first opening 4 is preferably 10-50 μm, the diameter of secondopening 5 is 1-5 μm, and the maximum diameter of hollow section 6 isaccordingly determined to be an optimal value between 10 μm and 100 μm.

[0060] As shown in FIG. 3, detecting electrode 7 made of gold is formedat least on an inner wall of second opening 5 and a lower part of hollowsection 6, and leader electrode 8 made of gold is provided on the lowersurface of substrate 1. Electrode 7 is electrically connected toelectrode 8 at second opening 5. No conductive material is provided onan upper part of hollow section 6, so that detecting electrode 7 iselectrically insulated from well 2.

[0061] Usage of the measuring device will be described below. FIG. 4 isa sectional view of the device having well 2 containing test cell 9 andculture solution 10 put thereinto. FIG. 5 through FIG. 9 are enlargedsectional views of first opening 4, second opening 5 and hollow section6.

[0062] As shown in FIGS. 4 and 5, just after putting culture solution 10and test cells 9 into well 2, cells 9 float in solution 10. Not onlywell 2 but also first opening 4, hollow section 6, and second opening 5are filled with solution 10, and then, solution overflows from secondopening 5. At this moment, as shown in FIG. 6, floating cell 9 is sackedonto first opening 4 by a pressure of solution 10 in well 2. If thepressure is small, solution 10 may be sucked with a suction pump throughsecond opening 5, thus allowing floating cell 9 to be sucked onto firstopening 4 more securely.

[0063] Since the diameter of first opening 4 is smaller than that ofcell 9, cell 9 receives a resistance when passing through opening 4, asshown in FIG. 7. However, since being forced by the pressure and thesuction, cell 9 can reach hollow section 6, while the cell deforms. Asshown in FIG. 8, cell 9 reaching hollow section 6 still receives thepressure of culture solution 10 from well 2 even if the suction stops.Since cell 9 has the diameter greater than that of opening 4, and sincefirst opening 4 is provided substantially vertically, cell 9 does notreturn to well 2 as long as an external force sucks cell 9 is notapplied from well 2. Thus, cell 9 is retained in hollow section 6.

[0064] Hollow section 6 has an oval shape, which has a lateral diametergreater than a vertical diameter smaller than the diameter of cell 9, asshown in FIGS. 7-9. These dimensions allow cell 9 to be held in hollowsection 6 without fail. At thus moment, chemicals (not shown) are dopedinto culture solution 10 in well 2 and permeate into solution 10. Thechemicals activate test cell 9, as shown in FIG. 9, and have the cell 9generate an electric signal at second opening 5. The electric signalchanges an electric potential of solution 10 at second opening 5. Thischange of the potential is detected by detecting electrode 7 and leaderelectrode 8 both contacting solution 10.

[0065] As such, the measuring device in accordance with Embodiment 1includes detecting electrode 7 electrically insulated from well 2, andhollow section 6 retains test cell 9 securely. In other words, culturesolution 10 at second opening 5 is electrically insulated from solution10 in well 2. Therefore, the electric signal generated through theactivities of the cell does not leak to solution 10 in well 2, and isdetected by detecting electrode 7 provided on second opening 5.

[0066] If any one of trap holes retains the test cell, an extracellularpotential can be measured.

[0067] First opening 4 may have a tapered shape flaring towards well 2,as shown in FIG. 29. This shape allows test cell 9 to enter into opening4 from well 2 easily. If a diameter of opening 4 at a boundary ofopening 4 against hollow section 6 is smaller than the maximum diameterof hollow section 6, test cell 9 is prevented from returning to well 2.Test cell 9 is thus trapped in hollow section 6, and the measuringdevice has a high retention rate of the cell. In this device, thediameter of hollow section 6 is greater than the diameter of firstopening 4 at the boundary against hollow section 6, and the diameter offirst opening 4 at the boundary against hollow section 6 is greater thanthe diameter of second opening 5.

[0068] A diameter of first opening 4 at a boundary against well 2 may besmaller than twice the diameter of the test cell, thus preventing pluralcells from entering into opening 4 simultaneously and from cloggingopening 4.

[0069] In the measuring device in accordance with Embodiment 1, whenonce entering into trap hole 101, test cell 9 cannot return to well 2.Thus, test cell 9, a somatic sample, contaminates well 2 and trap-hole101 during the measurement. The measuring device may be disposable andnot re-used, thus allowing cell 9 not to be took out.

[0070] Next, processes for manufacturing the measuring device inaccordance with Embodiment 1 will be described below. FIG. 10 throughFIG. 19 are sectional views of the measuring device which illustrate theprocesses.

[0071] First, as shown in FIG. 10, resist mask 11 is provided on siliconsubstrate 1 by a photo-lithography method in order to form well 2. Then,as shown in FIG. 11, well 2 is formed by etching substrate 1 upto apredetermined depth by a wet etching method or a dry etching method. Thewet etching method may employ KOH or tetramethyl ammonium hydroxide(TMAH) as etching solution. The dry etching method may employ SF₆ or CF₄as etching gas.

[0072] Then, as shown in FIG. 12, resist mask 12 for forming firstopening 4 is provided on a bottom of well 2, and resist mask 13 forforming second opening 5 is provided on a lower surface of siliconsubstrate 1. The diameters of openings 4 and 5 are determined accordingto a size of test cell 9. The diameter of first opening 4 is greaterthan that of second opening 5.

[0073] Next, as shown in FIG. 13, substrate 1 is etched from well 2 upto a predetermined depth for forming first opening 4. Substrate 1 may beetched preferably by a dry etching method employing bothetching-accelerator gas and etching-suppressor gas. The accelerator gasmay be SF₆ or CF₄ accelerating an etching of silicon not only depth wisebut also lateral wise. The gas may be mixed with CHF₃ or C₄F₈, whichsuppresses the etching, and forms a protective film made of polymer ofCF₂ on the wall of the opening, thus allowing the substrate to be etchedonly below the mask.

[0074] In order to etch the substrate in a vertical direction, thefollowing steps are repeated. That is, the substrate is etched a littlewith the etching-accelerator gas, and then, the protective film isformed with the etching-suppressor gas. These steps forms the openingsubstantially vertically. According to an experiment, first opening 4having a diameter of 20 μm by the following steps. SF₆ flows at a rateof 130 sccm to generate plasma for 13 seconds, thereby etching substrate1 by 1 μm. Then, C₄F₈ flows at a rate of 85 sccm to generate plasma for7 seconds, thereby forming the protective film having a thickness of0.01 μm. The steps of etching substrate 1 and forming the protectivefilm are repeated about 60 times, thereby forming a substantiallyvertical opening having a depth of 60 μm.

[0075] The protective film is formed not only on the wall of firstopening 4 but also on the bottom with the etching-suppressor gas. Theprotective film formed on the bottom can be removed by theetching-accelerator gas more easily than the protective film on thewall, thus allowing the substrate to be etched only downward.

[0076] First opening 4 is thus formed, while the protective film isformed with the etching-suppressor gas. After first opening 4 is formed,the protective film is formed on the wall of opening 4. The filmprotects the wall of opening 4 from damage during forming of hollowsection 6.

[0077] Then, as shown in FIG. 14, substrate 1 is etched from its lowersurface in order to form second opening 5. The etching-accelerator gasand the etching-suppressor gas are alternately used similarly to theforming of first opening 4, thus allowing the wall of second opening 5to be formed substantially vertically.

[0078] Further, the protective film is formed with theetching-suppressor gas, similarly to the forming of first opening 4, tocomplete the forming of second opening 5. The wall of opening 5 is thusprotected by the film securely, thus being prevented from damage whenhollow section 6 is formed in later processes.

[0079] Next, as shown in FIG. 15, substrate 1 is etched from firstopening 4 only with the etching-suppressor gas. The protective film isprovided on the wall of opening 4 at the previous process, thus allowingthe substrate to be etched downward without damage to the wall. Aportion which is newly etched does not have a protective film thereon,thus being etched also laterally. This etching forms hollow section 6which is provided between first opening 4 and second opening 5 and iswider than first opening 4, as shown in FIG. 15. Appropriate amount ofsubstrate 1 is etched to have hollow section 6 shaped in the oval havingthe lateral diameter greater than the vertical diameter.

[0080] After hollow section 6 communicates with second opening 5, theprotective film is still formed on the wall of opening 5. The filmprotects the wall of opening 5 from damage even though substrate 1 isbeing etched for a while until hollow section 6 has a predeterminedsize. If the substrate is excessively etched, hollow section 6 expandsnot only in a lateral direction but also in all directions, as denotedby dotted lines shown in FIG. 15. The substrate is finishedappropriately to etch.

[0081] The etching accelerator gas used in the above-described etchingmay include SF₆ or CF₄ and, however, preferably includes XeF₂ whichhardly etches the protective film. The gas of XeF₂ forms hollow section6 with little damages on the wall. The gas of XeF₂, however, needs along time to etch the protective film on the bottom of the openingformed in the previous process. In order to overcome this problem, theprotective film on the bottom may be etched with the gas, such as SF₆,CF₄ or Ar, before the gas of XeF₂ is used.

[0082] According to Embodiment 1, first opening 4, second opening 5 andhollow section 6 are formed in this order. However, second opening 5,first opening 4 and hollow section 6 may be formed in this order, orfirst opening 4, hollow section 6 and second opening 5 may formed inthis order. Hollow section 6 may be etched from second opening 5. Inthis case, the substrate is etched carefully to allow hollow section 6to be greater than first opening 4.

[0083] Next, as shown in FIG. 16, all the resist masks are removed, andthen, gold particles 14 are attached onto the wall by a vapor depositionmethod, thereby forming detecting electrode 7. In this process, goldparticles 14 are discharged from a target of first opening 4. Particles14 discharged from the target run straight, thus passing through firstopening 4. Then, as shown in FIG. 17, the particles deposit only on aninner wall of opening 4, a lower part of hollow section 6, and an innerwall of opening 5. In other words, detecting electrode 7 is formed onlyon the inner wall of second opening 5 and the lower part of hollowsection 6.

[0084] Then, as shown in FIG. 18, leading electrode 8 made from gold isformed on a surface of the substrate at second opening 5. Since secondopening 5 has a diameter smaller than that of first opening 4, goldparticles 15 run straight and deposit only on the inner wall of opening5 and a portion of the inner wall of opening 4. As shown in FIG. 19,detecting electrode 7 formed on the lower part of hollow section 6 andthe inner wall of second opening 5 is electrically insulated from thegold provided on the inner wall of first opening 4. In order to attachthe gold securely to substrate 1, a buffer layer of chrome or titan maybe provided on substrate 1, and the gold can be attached on the bufferlayer. In order to avoid depositing the gold on the bottom of well 2,the gold is deposited before resist mask 11, which has been disposed forforming first opening 4, is removed. The mask prevents the gold fromdepositing on the bottom of well 2 after resist mask 11 is removed. Thegold may be deposited by a sputtering instead of the vapor-deposition.

[0085] The second openings of the trap-holes have conductors formed onthe walls of the openings, and the conductors are short-circuited witheach other at the lower surface below the well. This structure createsparallel connection of electric potential changes around the test cellsheld in the trap-holes, so that the change in the electric potential ofeach test cell may be detected even if each electric potential change issmall.

[0086] The manufacturing method in accordance with Embodiment 1 allowssilicon substrate 1 to have well 2 and first opening 4, second opening5, and hollow section 6 which retains the test cell securely, thusproviding a reliable device for measuring an extracellular potential.

[0087] According to Embodiment 1, substrate 1 is made from silicon andhowever, may be made of material which can be dry-etched easily to beetched straight and laterally through switching etching gases. Forinstance, glass and quartz can be etched in a depth direction with gas,such as SF₆ or CF₄, and in a lateral direction with gas of HF.

[0088] According to Embodiment 1, first opening 4 is providedsubstantially perpendicularly to the bottom of well 2. First opening 4,upon having a tapered shape having a diameter at well 2 greater thanthat at hollow section 6, may be formed by the following processes. Whenthe gas including the etching-accelerator gas and the etching suppressorgas mixed is used, a concentration of the etching-accelerator gas isreduced according to proceeding of the etching from well 2 toward hollowsection 6. This operation allows the wall of opening 4 to taper, asshown in FIG. 20. This tapered shape allows test cell 9 to enter intoopening 4 easily, and prevents cell 9 once trapped in hollow section 6from return to well 2 easily.

[0089] In order to make the wall of opening 4 taper, substrate 1 may beetched with only the etching-accelerator gas. In this case, as shown inFIG. 22, the diameter of opening 4 at the boundary against well 2becomes greater than the diameter defined by resist mask 12. Therefore,the diameter defined by resist mark 12 is determined in advance in orderto get an optimum taper shape.

[0090] According to Embodiment 1, the relation among the diameters ofopenings 4 and 7 and hollow section 6 is described. As shown in FIG. 3,connecting portion 102, which is a border between opening 4 and hollowsection 6, has a diameter smaller than the maximum diameter of hollowsection 6. Connecting portion 103, which is a border between opening 7and hollow section 6, has a diameter smaller than that of connectingportion 102. This arrangement provides advantages identical to thosediscussed above.

[0091] Exemplary Embodiment 2

[0092]FIG. 23 is a perspective view of a device for measuring anextracellular potential in accordance with Exemplary Embodiment 2 of thepresent invention. FIG. 24 is a sectional view of the device. FIG. 25 isan enlarged sectional view of the device. Substrate 16 is formed bystacking first silicon layer 17, silicon dioxide layer 18, and secondsilicon layer 19, differently from a device of Embodiment 1. Firstopening 21, second opening 22, and hollow section 23 are formed in firstsilicon layer 17, second silicon layer 19, and silicon dioxide layer 18,respectively.

[0093] Detecting electrode 24 is formed only on an inner wall of secondopening 22 and a lower portion of hollow section 23, and leadingelectrode 25 is formed on a lower surface of substrate 16. Detectingelectrode 24 is electrically connected to leading electrode 25 aroundsecond opening 22.

[0094] An operation of the device discussed above is identical to thatof Embodiment 1, and the description thereof is thus omitted. Silicondioxide layer 18 between first silicon layer 17 and second silicon layer19 increases electrical insulation between the layers. Therefore, anelectric signal generated by activity of a cell at second opening 22 canbe detected securely by detecting electrode 24, and the signal does notleak to first opening 21.

[0095] Processes for manufacturing the device in accordance withEmbodiment 2 are described below. Description of processes identical tothose of Embodiment 1 is omitted, and only processes for forming firstopening 21, second opening 22, and hollow section 23 will be describedbelow. The substrate includes the silicon layer, the silicon dioxidelayer, and the silicon layer stacked in this order, which is availablein market as an SOI substrate, and is not thus explained.

[0096] First, well 20 is formed in first silicon layer 17, and then, asshown in FIG. 26, resist masks 26 and 27 is provided in order to formfirst opening 21 and second opening 22. Next, as shown in FIG. 27,layers 17 and 19 are dry-etched from a bottom of well 20 and a lowersurface of substrate 16, respectively, so that respective walls of theopenings become perpendicular to the bottom of well 20, and the openingsreach silicon dioxide layer 18. The substrate is etched, similarly toEmbodiment 1, with etching-accelerator gas for facilitating the etchingand etching-suppressor gas for suppressing the etching. In order to formopenings 21 and 22, layers 17 and 19 are etched until the openings reachsilicon dioxide layer 18. This etching requires no monitoring of anetching time for obtaining a predetermined depth.

[0097] Next, the substrate is dipped into solution of HF, which mainlyetches silicon dioxide layer 18 and etches layer 17 and 19 little toform hollow section 23, as shown in FIG. 28. Layer 18 is etched untilhollow section 23 has a necessary lateral diameter. Then, similarly toEmbodiment 1, detecting electrode 24 and leading electrode 25 areformed. Layer 18 may be etched with plasma using HF gas, which etchesthe silicon layers little but etches mainly silicon dioxide layer 18similarly to the HF solution. An etching of Embodiment 1, an etching ofexcessively long time does not make the hollow section oval; however,the method of Embodiment 2 overcomes this problem.

[0098] In substrate 16 including two kinds of layers, namely, siliconand silicon dioxide layers, the depth of second opening 22 and theheight of hollow section 23 are determined in advance, thus allowing thedevice to be manufactured easily. Silicon dioxide layer 18 completelyisolates first opening 21 electrically from second opening 22, thusproviding a reliable measuring device.

[0099] According to Embodiment 2, substrate 16 includes three layers,i.e., first silicon layer 17, silicon dioxide layer 18, and secondsilicon layer 19. However, the substrate may include four layers, i.e.,a silicon layer, a silicon dioxide layer, a silicon layer, and a silicondioxide layer, or more than four layers.

[0100] Substrate 16 is formed by stacking the silicon layer, the silicondioxide layer, and the silicon layer in this order. However, a substrateformed by stacking a silicon dioxide layer, a silicon layer, and asilicon dioxide layer in this order may provides the device. Substrate16 may be made of not only the combination of silicon and silicondioxide, but also other combinations, such as silicon and glass,aluminum and aluminum oxide, or glass and resin. Substrate 16 may bemade of three materials instead of the two materials, and may includelayers of materials different from each other. Such substrates providesadvantages similar to those discussed above.

[0101] Similarly to Embodiment 1, as shown in FIG. 3, a first connectionsection at a border between the first opening and the hollow section hasa diameter smaller than the maximum diameter of the hollow section, anda second connection section at a border between the second opening andthe hollow section has a diameter smaller than that of the firstconnection section. This structure provides advantages similar to thoseof Embodiment 2.

[0102] Exemplary Embodiment 3

[0103]FIG. 30 is a perspective view of a device for measuring anextracellular potential in accordance with Exemplary Embodiment 3 of thepresent invention. FIGS. 31A, 31B and 32 are sectional views of thedevice. FIGS. 33 and 34 are enlarged sectional views of the device. FIG.35 through FIG. 42 are sectional views of the device for illustrating amethod of manufacturing the device.

[0104] As shown in FIG. 30 through FIG. 32, substrate 28 has a laminatedstructure including base 29 made of silicon, intermediate layer 30 madeof silicon dioxide, and thin plate 31 made of silicon. Base 29 has well32 therein for accommodating sample solution including test cells. Well32 is used for mixing test cells 37 and culture solution with chemicals.Further, thin plate 31 forming the bottom of well 32 has through-holes33 therein. Well 32 has pockets 34 formed at holes 33, and thus, adiameter of hole 33 at well 32 is greater than a diameter of hole 33 ata lower surface of substrate 28.

[0105] Diameters of through-holes 33 and pockets 34 may be determinedaccording to a size and characteristics of test cell 37. For the cell 37having a diameter of 10 μm, the pocket 34 having a diameter ranging from10 μm to 20 μm and the hole 33 having a diameter smaller than 5 μm aresuitable.

[0106] According to Embodiment 3, an inner wall of pocket 34 has aconical shape having its bottom towards well 32.

[0107] Insulator 36 made from silicon dioxide is provided on the innerwall and the bottom of well 32, the inner wall of through-hole 33, theinner wall of pocket 34, and the lower surface of thin plate 31.Detecting electrodes 35 made mainly of gold are provided on a portion ofinsulator 36 on the inner wall of hole 32 and the outside of thin plate31.

[0108] The cell generally has a diameter of 5 μm-20 μm, and thus, anopening of pocket 34 preferably has a diameter of 10 μm-100 μm, and anopening of hole 33 has a diameter of 1 μm-10 μm. The device discussedabove can measure an extracellular potential, i.e., a physico-chemicalchange generated by the cell by the following operation described belowwith reference to figures.

[0109]FIG. 32 is a sectional view of well 32 having test cell 37 andculture solution 38 put therein. FIGS. 33 and 34 are enlarged sectionalviews of an essential portion including through-hole 33 and pocket 34.As shown in FIG. 32, just after culture solution 38 and test cell 37 areinput in well 32, cell 37 floats in solution 38. Well 32 is filled withsolution 38 as well as pocket 34 and hole 33 are filled with solution38, and then, solution 38 overflows to the lower surface of well 32. Asshown in FIG. 33, this flow allows floating cell 37 to be sucked inpocket 34 by a pressure of solution 38 in well 32. If the pressure issmall, solution 38 may be sucked with a suction pump from hole 33 toallow floating cell 37 to be sucked more securely in pocket 34.

[0110] Next, test cell 37 reaching pocket 34 receives a pressure bysuction from hole 33 or by culture solution 38 from well 32, thus beingretained in pocket 34, as shown in FIG. 33. At this moment, chemicals(not shown) may be doped into culture solution 38 in well 32 to permeateinto solution 38. When the chemicals activates test cell 37 due toreaction by ion-exchange, as shown in FIG. 34, an electric signalgenerated in hole 33 changes an electric potential of a portion ofculture solution 38 filled in hole 33. This electric potential change isdetected by detecting electrode 35 contacting solution 38.

[0111] As described above, pocket 34 provided in the bottom of well 32allows the device in accordance with Embodiment 3 not to require anotherwell. Test cell 37 and the culture solution can be mixed with thechemicals in well 32. Well 32, pockets 34 provided in the bottom, andtrough-holes 33 are unitarily formed, thus preventing culture solution38 from leaking outside well 32 by mistake, and thus allowing thesolution to flow to aperture 33.

[0112] Insulator 36 of silicon dioxide provided on the inner wall ofpocket 34, the inner wall of through-hole 33, the lower surface of thinplate 31, the bottom, and the inner wall of well 32 electricallyinsulates detecting electrode 35 from well 32. Since pocket 34 has theconical shape having its bottom towards well 32, cell 37 is sucked intopocket 34 and is retained in the pocket stably, thus preventing cell 37from staying in aperture 33. For instance, if test cell 37 has adiameter of 10 μm, the diameter of pocket 34 at well 32, namely, thebottom of the conical shape is determined to be less than 20 μm, thusplural cells 37 not to enter into pocket 34 at once. Through-hole 33having a diameter less than 5 μm does not allow cell 37 to pass throughhole 33.

[0113] As discussed above, test cell 37 can be securely retained inpocket 34 during measuring. A portion of culture solution 38 in hole 33is electrically insulated from a portion of solution 38 in well 32, thuspreventing the electric signal generated by activity of test cell 37from leaking to the portion of solution 38 in well 32. Therefore, thesignal is detected by detecting electrode 35 provided on hole 33.Insulator 36 is necessary when a surface layer of the silicon substratehas so a small resistivity. Alternatively, insulator 36 is necessarywhen the electric signal generated in hole 33 is too weak to be measureddue to a little leak of the electric signal to well 32.

[0114] Therefore, if the silicon substrate has a large surfaceresistivity, test cell 37 that is retained assures enough electricinsulation. Therefore, when an extracellular potential is large enoughnot to be influenced by a little leakage of the electric signal,insulator 36 is not necessarily required.

[0115] Next, a method of manufacturing the device in accordance withEmbodiment 3 will be described below with reference to FIG. 35 to FIG.42. First, as shown in FIG. 35, substrate 38 including base 29 made ofsilicon, intermediate layer 30 made of silicon dioxide, and thin plate31 made of silicon is prepared. Resist mask 39 is provided on the lowersurface of thin plate 31. Substrate 28 may be an SOI substrate, which isoften used for manufacturing semiconductor devices. The SOI substrate isavailable in market, and thus, a method for manufacturing the substrateis not described.

[0116] Then, thin plate 31 is dry-etched to form through-hole 33 havinga predetermined depth. FIG. 36 is an enlarged of portion A in FIG. 35.It is important that the plate is preferably dry-etched withetching-accelerator gas for facilitating the etching andetching-suppressor gas for suppressing the etching, similarly toEmbodiment 1. These gases allow through-hole 33 to be formed onlybeneath resist mark 39, as shown in FIG. 36.

[0117] While substrate 28 is dry-etched with the etching-accelerator gasand the etching-suppressor gas used alternately, a high frequency isapplied to substrate 28, and an inductive-coupling method with anexternal coil is used for the etching. The high frequency generates anegative bias potential in substrate 28 and makes positive ions inplasma, such as SF₅ ⁺ or CF₃ ⁺ collide with substrate 28, thus allowssubstrate 28 to be etched perpendicularly to the bottom.

[0118] The dry-etching may be suppressed by stopping applying the highfrequency to substrate 28. The bias potential is stopped, and CF₊,material of a protective film, is not deflected. As a result, theprotective film is formed uniformly on the walls of the through-holes insubstrate 28.

[0119] The method described above is effectively applicable to formingan opening perpendicular to the bottom of the substrate by manufacturingmethods described in Embodiments 1 and 2.

[0120] Next, as shown in FIG. 37, thin plate 31 is dry-etched until thehole reaches intermediate layer 30. In this process, a concentration ofthe etching-accelerator gas is gradually increased according to theprogress of the etching toward the bottom of well 32. Alternatively, atime of the etching with the etching-accelerator gas is graduallyincreased. In other words, when facilitating of the dry-etching andsuppressing of the dry-etching are alternately repeated, the ratio of atime of the facilitating to a time of the suppressing is graduallyincreased.

[0121] This operation allows the hole 33 to flaring toward well 32, asshown in FIG. 37, thus allowing through-hole 33 to communicate withpocket 34 flaring toward well 32. When the dry-etching is finished, i.e.when hole 33 reaches intermediate layer 30, the dry-etching is notnecessarily stopped immediately due to intermediate layer 30 made fromsilicon dioxide since the etching gas does not etch intermediate layer30 immediately.

[0122] The etching-accelerator gas of SF₆ cannot easily dry-etchintermediate layer 30 made of silicon dioxide to remove the intermediatelayer since a ratio of an etching rate for silicon to that for silicondioxide is more than ten. Therefore, even if the dry-etching continuesfor a while after the hole reaches the silicon dioxide layer, thedry-etching can hardly remove layer 30, thus forming pocket 34accurately and easily.

[0123] Then, as shown in FIG. 38, resist mask 40 is provided on base 29by a photo-lithography method. Then, as shown in FIG. 39, base 29 isetched until well 5 reaches intermediate layer 30. At this process, theetching-accelerator gas and the etching-suppressor gas may be used, aspreviously discussed, for providing wells 5 at a high density. However,if the high density is not needed, a wet-etching using TMAH or KOH isacceptable.

[0124] Next, as shown in FIG. 40, a portion of intermediate layer 30made of silicon dioxide exposed from the bottom of well 32 is removed bya wet-etching using HF or a dry-etching with CF₄ gas.

[0125] Then, as shown in FIG. 41, a silicon-dioxide layer is formed onthe surface of the silicon substrate, which includes base 29 and thinplate 31, by a thermal oxidation method. This process providesinsulating layer 36 made from silicon dioxide on the inner wall and thebottom of well 32, the inner wall of pocket 34, the inner wall of hole33, and the lower surface of thin plate 31.

[0126] Next, as shown in FIG. 42, detecting electrode 35 is formed onthe lower surface of thin plate 31 by vapor-depositing or sputteringgold. Thus, electrode 35 is formed not only on the lower surface ofplate 31 but also on the inner wall of through-hole 33. Electrode 35 ismade of material not to react on culture solution 38. The material maybe preferably selected from gold, platinum, silver, silver chloride, andaluminum appropriately according to a type of the sample solution.

[0127] The method in accordance with Embodiment 3 described aboveprovides the device having through-holes 33 in thin plate 31 and conicalpockets 34 communicating with holes 33 to well 34 accurately and easilyby a one-time etching.

[0128] In the method of Embodiment 3, it is not necessary that thesubstrate is etched with two kinds of resist masks by thephotolithography method from the well. This method allows through-holesand pockets to be formed accurately in the bottom of the well even ifthe substrate has bumps and dips therein. Even the substrate having suchrough surface may have the through-holes and the pockets in the bottomof the well accurately without using a spray-coating device which coatthe rough surface uniformly with a resist mask, or using a projection ora stepper forming a highly accurate pattern onto the resist mask byexposure to light with non contact between a photo mask and thesubstrate.

[0129] Exemplary Embodiment 4

[0130]FIG. 43 is a sectional view of a device for measuring anextracellular potential in accordance with Exemplary Embodiment 5 of thepresent invention. FIGS. 44 and 45 are enlarged sectional views ofessential portions of the device.

[0131] The device in accordance with Embodiment 5 has a structurebasically identical to that of Embodiment 3, and thus, similar elementsare not described.

[0132]FIG. 43 is a sectional view of the device in accordance withEmbodiment 5. Pocket 47 formed in thin plate 44 has a hemisphere shape,which retains test cell 37 more closely thereto, so that a change of anelectrical potential of culture solution 38 in through-hole 46 can bedetected more easily.

[0133] A method of manufacturing the device will be described below. Themethod in accordance with Embodiment 4 is similar to that of Embodiment3. According to Embodiment 4, through-hole 46 and pocket 47 is formed inthin plate 44 by a method different from that of Embodiment 3. Thedifferent method will be described with reference to FIGS. 44 and 45.

[0134] As shown in FIG. 44, resist mask 50 is formed on thin plate 44while intermediate layer 43 and thin plate 44 are attached to eachother. Then, thin plate 44 is dry-etched with etching-accelerator gasfor facilitating the etching and etching-suppressor gas for suppressingthe etching up to a predetermined depth to form through-hole 46. Thepredetermined depth is determined to prevent the etching from reachingintermediate layer 43 made of silicon dioxide, and determined to be anoptimum depth in response to a size and a shape of pocket 47.

[0135] During this etching, thin plate 44 is etched with theetching-accelerator gas, and then has a protective film (not shown)formed thereon with the etching-suppressor gas. These processes fordry-etching thin plate 44 are repeated perpendicularly to resist mask 50and only under an opening of resist mask 50. These processes terminateby dry-etching thin plate 44 with the etching-accelerator gas. Thisoperation removes the protective film formed by the etching-suppressorgas from the bottom of the etched place.

[0136] Next, as shown in FIG. 45, pocket 47 is formed by dry-etchingwith XeF₂ gas. The dry-etching progresses from the bottom where siliconis exposed, and corroded area becomes greater as the etching progressestoward well 45. The inner wall of through-hole 46 has the protectivefilm formed thereon by the etching-suppressor gas, so that the wall ofhole 46 is not dry-etched by the XeF₂ gas. Pocket 47 thus has ahemisphere shape, as shown in FIG. 45 shows. After these processesdiscussed above, the substrate undergoes the processes shown in FIG. 38through FIG. 42 similarly to that of Embodiment 3, thereby providing thedevice for measuring an extracellular potential.

[0137] Exemplary Embodiment 5

[0138] According to Exemplary Embodiment 5, another method for formingthrough-hole 33 and 46 and pocket 34 and 47 described in Embodiments 3and 4, respectively, will be described.

[0139] The method of forming the through-hole different from the methodsof Embodiments 3 and 4 will be described hereinafter with reference toFIG. 46 through FIG. 48.

[0140]FIG. 46 is a sectional view of a measuring device in accordancewith Embodiment 5 for illustrating a method of manufacturing the device.Intermediate layer 51 made of silicon dioxide and thin plate 52 made ofsilicon are stacked on each other. Then, resist mask 53 is provided onthin plate 52, and through-hole 56 is formed by dry-etching a substratewith etching-accelerator gas for facilitating the etching andetching-suppressor gas for suppressing the etching. Thin plate 44 iscontinued to etch until hole 56 reaches intermediate layer 51.

[0141] Thin plate is still continued to etch after hole 56 reachesintermediate layer 51 which is made of insulator and has a resistancesmaller than thin plate 52 made of silicon. As shown in FIG. 47,excessive etching makes etching ions 54, such as SF₅ ⁺, stay on thesurface of layer 51 when the etching-accelerator gas is used for thedry-etching, so that etching ions 55 supplied from plasma deflects alongan arrow shown in FIG. 47.

[0142] As a result, the vicinity of the wall of intermediate layer 51 islocally dry-etched, and through-hole 56 flares toward the well, namely,pocket 57 is formed, as shown in FIG. 48.

[0143] According to an experiment, thin plate 44 is continued todry-etch after hole 56 having a diameter of 3 μm reaches intermediatelayer 51, thereby providing pocket 57 having a maximum diameter of 10μm.

INDUSTRIAL APPLICABILITY

[0144] A device for measuring an extracellular electric potentialaccording to the present invention allows a test cell to enter in ahollow section of the device. When the test cell once enters in thehollow section, the cell is trapped therein securely. The device thuscan detect an electric signal generated by activities of the cellwithout fail.

1. A device for measuring an extracellular potential of a test cell,said device comprising: a substrate having a well formed in a firstsurface thereof and a first trap hole formed therein, the well having abottom, the first trap hole including a first opening formed in thebottom of the well and extending toward a second face of the substrate,a first hollow section communicating with the first opening via a firstconnecting portion, and a second opening extending reaching the secondsurface and communicating with the first hollow section via a secondconnecting portion, wherein the first connecting portion has a diametersmaller than a maximum diameter of the first hollow section, greaterthan a diameter of the second connecting portion, and smaller than adiameter of the test cell.
 2. The device of claim 1, wherein the firstopening and the second opening are aligned on a straight line.
 3. Thedevice of claim 1, wherein the substrate includes silicon.
 4. The deviceof claim 1, wherein the substrate comprising first and second layersstacked on each other, the first layer being made of material having anetching rate different from an etching rate of material of the secondlayer.
 5. The device of claim 4, wherein the first layer includessilicon, and the second layer includes silicon dioxide.
 6. The device ofclaim 4, wherein one of the first and second openings is formed in thefirst layer, and the first hollow section is formed in the second layer.7. The device of claim 1, further comprising a first conductive layerformed on a wall of the second opening and a portion of a wall of thefirst hollow section connected to the second connecting portion.
 8. Thedevice of claim 7, wherein the first conductive layer does not reach thefirst connecting portion.
 9. The device of claim 1, wherein the firstopening flares toward the well from the first connecting portion. 10.The device of claim 9, wherein the first opening has, at the bottom ofthe well, a diameter smaller than twice the diameter of the test cell.11. The device of claim 1, wherein the first hollow section has a firstdiameter along a direction from the first connecting portion to thesecond connecting portion and a second diameter perpendicular tocrossing the direction of the first diameter, and wherein the seconddiameter is greater than the first diameter, and the first diameter issmaller than the diameter of the test cell.
 12. The device of claim 11,wherein the diameter of the first connecting portion ranges from 10 μmto 50 μm, wherein the diameter of the second connecting portion rangesfrom 1 μm to 5 μm, and wherein the second diameter of the first hollowsection ranges from 10 μm to 100 μm, and the first diameter of the firsthollow section is not more than 50 μm.
 13. The device of claim 1,wherein the substrate has a second trap hole including a third openingformed in the bottom of the well and extending toward the second surfaceof the substrate, a second hollow section communicating with the thirdopening via a third connecting portion, and a fourth openingcommunicating with to the second hollow section via a fourth connectingportion and extending to the second surface of the substrate, andwherein the third connecting portion has a diameter smaller than amaximum diameter of the second hollow section, greater than a diameterof the third connecting portion, and smaller than the diameter of thetest cell.
 14. The device of claim 13, further comprising a secondconductive layer formed on a wall of the fourth opening and a portion ofa wall of the second hollow section connected to the fourth connectingportion, wherein the second conductive layer does not reach the thirdconnecting portion.
 15. The device of claim 14, further comprising athird conductive layer formed on the second surface of the substrate,for connecting the first conductive layer to the second conductivelayer.
 16. A method of manufacturing a device for measuring anextracellular potential of a test cell, said method comprising the stepsof: forming a well having a bottom in a first surface of a substrate;providing a first mask having a first hole on the bottom of the well;forming a first opening through the first hole of the first mask bydry-etching with first gas for suppressing etching and second gas forfacilitating the etching; providing a second mask having a second holeon a second surface of the substrate; forming a second opening from thesecond hole of the second mask towards the first opening by dry-etchingthe substrate with the first gas and the second gas; and forming ahollow section between the first opening and the second opening byetching the substrate with the second gas.
 17. The method of claim 16,wherein said step of providing the second mask is executed after saidstep of forming the first opening.
 18. The method of claim 17, whereinsaid step of forming the hollow section comprises forming the hollowsection by etching the substrate with the second gas from the secondopening.
 19. The method of claim 16, wherein the second gas is one ofSF₆, CF₄, XeF₂, and or a mixture thereof, and the first gas is one ofCHF₃, C4F₈, and a mixture thereof.
 20. The method of claim 16, whereinsaid step of forming the first opening comprises reducing a ratio of aconcentration of the second gas to a concentration of the first gas asthe substrate is etched from the well toward the hollow section.
 21. Themethod of claim 16, wherein said step of forming the first openingcomprises forming the first opening by dry-etching the substrate withusing the first gas and the second gas alternately.
 22. The method ofclaim 16, wherein said step of forming the first opening terminates bydry-etching the substrate with the first gas.
 23. The method of claim16, wherein said step of forming the first opening comprises formingproduct generated by dry-etching the substrate with the first gas on awall of the first opening.
 24. The method of claim 16, wherein said stepof providing the first mask is executed after said step of forming thesecond opening.
 25. The method of claim 24, wherein said step of formingthe hollow section comprises forming the hollow section by etching thesubstrate with the second gas from the first opening.
 26. The method ofclaim 16, further comprising the step of forming a conductive layer onthe second opening and a portion of the hollow section.
 27. The methodof claim 26, wherein said step of forming the conductive layer comprisesforming the conductive layer by vapor-depositing or sputteringconductive particles supplied through the first opening.
 28. A devicefor measuring an extracellular potential of a test cell, said devicecomprising: a substrate including a base, an intermediate layer stackedon the base and made of material different from material of the base,and a thin plate stacked on the intermediate layer and made of materialidentical to the material of the base, wherein the base and theintermediate layer has a well formed in the base and the intermediatelayer, the well having a bottom and extending from the base to theintermediate layer, wherein the thin plate has a through-holecommunicating with the well and an outside of the substrate.
 29. Thedevice of claim 28, wherein the through-hole includes a pocket at thewell, and an opening of the pocket has a diameter greater than adiameter of an opening of the through-hole.
 30. The device of claim 29,wherein the diameter of the opening of the pocket recess ranges from 10μm to 100 μm, and the diameter of the opening of the aperture rangesfrom 1 μm to 10 μm.
 31. The device of claim 29, wherein the pocket hasan inner wall having a conical shape.
 32. The device of claim 29,wherein the pocket has an inner wall having a hemisphere shape.
 33. Thedevice of claim 28, wherein the intermediate layer has an resistivitygreater than a resistivity of the thin plate.
 34. The device of claim28, wherein the material of the intermediate layer has an etching ratedifferent from an etching rate of the material of the thin plate. 35.The device of claim 28, wherein the base includes silicon, and theintermediate layer includes silicon dioxide.
 36. A method ofmanufacturing a device for measuring an extracellular potential of atest cell, said method comprising the steps of: providing a substratewhich includes a base, an intermediate layer stacked on the base andmade of material different from material of the base, and a thin platestacked on the intermediate layer and made of material identical to thematerial of the base; forming a well by etching the base such that aportion of the intermediate layer is exposed; forming a hole providedfrom the thin plate and reaching intermediate layer by dry-etching usingthe mask; and removing a portion of the exposed portion of theintermediate layer; and forming a detecting electrode on a wall of thehole from a side opposite to the intermediate layer.
 37. The method ofclaim 36, wherein said step of forming the hole comprises forming thehole by dry-etching a side opposite to the intermediate layer.
 38. Themethod of claim 36, wherein the intermediate layer includes silicondioxide.
 39. The method of claim 36, wherein said step of forming thehole comprises forming the hole by dry-etching with first gas forsuppressing etching and second gas for facilitating etching.
 40. Themethod of claim 39, wherein said step of forming the hole comprisesdry-etching the substrate with the first gas for a longer time as thedry-etching progresses, and dry-etching the substrate with the secondgas for a shorter time as the dry-etching progresses.
 41. The method ofclaim 39, wherein said step of providing the perforated apertureincludes a step of providing the aperture by the dry-etching with thefirst gas and the second gas alternately used.
 42. The method of claim41, wherein said step of forming the hole comprises forming the hole bythe dry-etching with using the first gas and the second gas alternatelybefore the hole reaches the intermediate layer, and then, forming thehole by dry-etching with the first gas.
 43. The method of claim 39,wherein said step of forming the hole comprises applying a highfrequency to the substrate during etching the substrate with the firstgas.
 44. The method of claim 39, further comprising the step ofdry-etching the thin plate after the hole reaches the intermediatelayer.
 45. The method of claim 39, wherein the first gas includes atleast one of SF₆, CF₄, and XeF₂.
 46. The method of claim 39, wherein thesecond gas includes at least one of C₄F₈ and CHF₃.